Synthesis of cyclic peptidotriazoles with activity against phytopathogenic bacteria

Article abstract of DOI:10.1002/ejoc.201300215

Cyclic peptidotriazoles derived from the antimicrobial cyclic peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln) (BPC194) were prepared by incorporating a triazolyl amino acid at the 3-position. The synthesis was accomplished on solid-phase and involved a

Full text of DOI:10.1002/ejoc.201300215

Job/Unit: O30215
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Pages: 12
FULL PAPER
Antibiotics
I. Güell, S. Vilà, L. Micaló, E. Badosa,
E. Montesinos, M. Planas,*
L. Feliu* ......................................... 1–12
Synthesis of Cyclic Peptidotriazoles with
Activity Against Phytopathogenic Bacteria
Keywords: Click chemistry
/ Cycload-
dition / Antibiotics / Peptides / Peptido-
mimetics
Cyclic peptidotriazoles were synthesized on
solid-phase from an alkyl or an azido pept-
idyl resin. Sequences active against phyto-
pathogenic bacteria and with low hemo-
lysis were identified.
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M. Planas, L. Feliu et al.
FULL PAPER
DOI: 10.1002/ejoc.201300215
Synthesis of Cyclic Peptidotriazoles with Activity Against Phytopathogenic
Bacteria
Imma Güell,[a] Sílvia Vilà,[a] Lluís Micaló,[a] Esther Badosa,[b] Emilio Montesinos,[b]
Marta Planas,*[a] and Lidia Feliu*[a]
Keywords: Click chemistry / Cycloaddition / Antibiotics / Peptides / Peptidomimetics
Cyclic peptidotriazoles derived from the antimicrobial cyclic
peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln)
linear peptidyl sequence linked through a triazolyl ring. Cy-
clic peptidotriazoles were obtained in excellent purities start-
ing either from an alkynyl or an azido peptidyl resin. These
compounds were screened in vitro for their growth inhibition
of bacterial phytopathogens and for their cytotoxic effects on
eukaryotic cells. Peptide sequences with high antibacterial
activity and low hemolysis were identified, constituting good
candidates for the design of new antimicrobial agents.
(BPC194) were prepared by incorporating a triazolyl amino
acid at the 3-position. The synthesis was accomplished on
solid-phase and involved as the key step a copper-catalyzed
cycloaddition reaction between a resin-bound alkyne or a
resin-bound azide and a range of azides or alkynes in solu-
tion, respectively. This methodology was also applied to the
synthesis of a conjugated peptide containing a cyclic and a
ties and because of the general synthetic method that is now
available.[4] In fact, there are a large number of compounds
that contain a 1,2,3-triazole ring with interesting biological
activities, such as anticancer, antimicrobial and antivirus
agents. Concerning the formation of this ring, copper-cata-
lyzed azide alkyne cycloaddition (CuAAC), first reported
by Meldal and co-workers, regioselectively provides 1,4-di-
substituted 1,2,3-triazoles.[4a,4c,4d,5] This reaction has been
used for the macrocyclization or derivatization of peptide
sequences, and as a method for linking peptide fragments,
allowing the synthesis of biologically active peptidotriaz-
oles.[4a,4b,5e]
The CuAAC has been successfully applied to the prepa-
ration of cyclic peptidotriazoles bearing the triazole ring in
the peptide backbone.[6] Although very promising, modifi-
cation of cyclic peptides by incorporating a triazole onto
the side-chain of a selected residue has rarely been reported
and has mainly been applied to obtain peptide conjugates.[7]
Moreover, despite the potential interest of this type of cyclic
peptidotriazoles, to the best of our knowledge their evalu-
ation as antimicrobial agents has not been described.
During our current research into the development of new
antimicrobial agents, we have identified cyclic decapeptides
Introduction
The vulnerability of modern agriculture to diseases and
pests has resulted in an intense research effort to develop
new antimicrobial agents that are safe for the host organism
and the environment, and that are unlikely to cause the
emergence of resistant strains. Due to their broad spectrum
of activity, low intrinsic cytotoxicity, and unique mecha-
nism of action, antimicrobial peptides are considered to be
suitable candidates for use in plant protection.[1]
Many synthetic antimicrobial peptides incorporating un-
natural amino acids and maintaining the structural features
of the natural sequences have been designed and have been
reported to exhibit comparable or improved biological pro-
files.[2] In particular, our research group has reported linear
undecapeptides incorporating a triazolyl amino acid de-
rived from the antimicrobial peptide BP100. These peptido-
triazoles displayed potent activity against plant pathogenic
bacteria and fungi, low hemolysis, and high stability
towards protease degradation.[3]
The 1,2,3-triazole ring has been increasingly used in drug
discovery because of its favorable physicochemical proper-
[a] Laboratori d’Innovació en Processos i Productes de Síntesi
Orgànica (LIPPSO), Departament de Química, Universitat de with high activity against the gram-negative plant patho-
Girona,
genic bacteria Xanthomonas axonopodis pv. vesicatoria, Er-
Campus Montilivi, 17071 Girona, Spain
winia amylovora, and Pseudomonas syringae pv. syringae.[8]
Fax: +34-972-418150
E-mail: marta.planas@udg.edu, lidia.feliu@udg.edu
Homepage: http://www.lippso.com
The most active peptide, c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-
Lys-Leu-Gln) (BPC194), also showed minimal cytotoxicity
and high stability to protease degradation. In this work,
we decided to study the solid-phase synthesis of BPC194
analogues incorporating a 1,2,3-triazole ring at the side-
chain of a selected residue and evaluate its influence on the
[b] Laboratory of Plant Pathology, Institute of Food and
Agricultural Technology-CIDSAV-Cer.t.A, Universitat de
Girona,
Campus Montilivi, 17071 Girona, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300215.
2
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Cyclic Peptidotriazoles Against Phytopathogenic Bacteria
biological activity. For this purpose, cyclic peptidotriazoles Leu3 for residues incorporating a triazole at the side-chain
I and II, bearing a triazolyl amino acid at the 3-position, (Figure 1). A Leu residue was selected because the cationic
were designed (Figure 1). Their synthesis was envisaged Lys residues are essential for the activity of these peptides.
from a resin-bound alkyne or a resin-bound azide, respec- Moreover, among the two Leu residues present in the se-
tively. Subsequently, we studied the antibacterial activity of quence, Leu3 was chosen to be replaced because the frag-
the synthesized cyclic peptidotriazoles against phytopathog- ment Lys5-Phe-Lys-Lys-Leu-Gln10 was proven to be a
enic bacteria and also their hemolytic activity.
structural requirement for high antibacterial activity.[8] In
particular, for cyclic peptidotriazoles I, Leu3 was substi-
tuted by an alanine, a glutamic acid or a lysine residue in-
corporating a 1,2,3-triazol-4-yl substituent at the side-
chain. For cyclic peptidotriazoles II, Leu3 was replaced with
a norleucine residue bearing a 1,2,3-triazol-1-yl substituent
at the side-chain. The triazole moiety was either unsubsti-
tuted or substituted with an alkyl, an aryl, or a peptidyl
group.
Synthesis of Cyclic Peptidotriazoles I
The synthesis of cyclic peptidotriazoles I was based on a
cycloaddition reaction between a resin-bound alkyne and
an azide in solution. According to this strategy, the alkynyl
cyclic peptidyl resins 1–3 were prepared (Scheme 1,
Scheme 2, and Scheme 3). Resin 1 incorporated a propar-
gylglycine at the 3-position whereas resin 2 contained a glu-
tamic acid substituted with a propargylamine, and resin 3
incorporated a lysine bearing a propioloyl group.
The general strategy for the solid-phase synthesis of the
alkynyl cyclic peptidyl resins 1–3 involved the preparation
of a linear peptidyl sequence followed by head-to-tail cycli-
zation. A three dimensional orthogonal 9-fluorenylmeth-
oxycarbonyl (Fmoc)/tert-butyl (tBu)/allyl (All) strategy was
used.[9] Thus, resin 1 was prepared starting from Fmoc-
Rink-MBHA (0.3 mmol/g) (Scheme 1). After Fmoc re-
moval by treatment with piperidine/N,N-dimethylform-
amide (DMF) (3:7), Fmoc-Glu-OAll was coupled to the
support by using ethyl 2-cyano-2-(hydroxyimino)acetate
(Oxyma) (4 equiv.) and N,N-diisopropylcarbodiimide
(DIPCDI) (4 equiv.) in DMF for 1 h. The linear sequence
was then elongated by sequential Fmoc removal and cou-
pling steps. Fmoc-Prg-OH was incorporated at the 3-posi-
tion. Once the protected linear sequence 4 was completed,
the allyl group was removed by treatment with [Pd-
(PPh3)4] (3 equiv.) in CHCl3/AcOH/N-methylmorpholine
(NMM) (3:2:1) for 3 h. Following Fmoc removal, cycliza-
tion was performed by using [ethyl cyano(hydroxyimino)-
acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophos-
phate (PyOxim) (5 equiv.), Oxyma (5 equiv.), and diisopro-
pylethylamine (DIPEA) (10 equiv.) in N-methyl-2-pyrrol-
idinone (NMP) for 24 h. An aliquot of resin 1 was subjected
to acidolytic cleavage with trifluoroacetic acid (TFA)/H2O/
triisopropylsilane (TIS) (95:2.5:2.5) for 2 h, affording
Figure 1. General structure of cyclic peptidotriazoles I and II.
Results and Discussion
peptide
c(Lys-Lys-Prg-Lys-Lys-Phe-Lys-Lys-Leu-Gln)
(BPC466), which was analyzed by HPLC (96% purity) and
characterized by mass spectrometry.
Design of the Cyclic Peptidotriazoles
Cyclic peptidotriazoles with general structure I and II
The synthesis of the alkynyl cyclic peptidyl resin 2
were designed based on the sequence of BPC194 c(Lys1- (Scheme 2) followed the same strategy described above for
Lys-Leu3-Lys-Lys-Phe-Lys-Lys-Leu-Gln10) by replacing resin 1. In this case, the preparation of the linear peptidyl
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Scheme 1. Synthesis of the alkynyl cyclic peptidyl resin 1.
resin 5 required the use of Fmoc-Glu(NH-CH2-CϵCH)-
Next, the alkynyl cyclic peptidyl resin 3, with Lys3
OH as alkynyl-functionalized residue. This amino acid is bearing a propioloyl group at the side-chain, was prepared
not commercially available but was easily prepared from by following the above procedure (Scheme 3). To achieve
Fmoc-Glu-OtBu by amidation of the side-chain carboxylic the selective acylation of Lys3, this residue was incorporated
acid group with propargylamine followed by hydrolysis of with the Nε-amino group protected with a 4-methyltrityl
the tBu ester. The amidation was achieved by treatment of (Mtt) group. This group is highly acid labile and can be
Fmoc-Glu-OtBu with propargylamine, N-(3-dimethylami- selectively removed without compromising the resin
nopropyl)-NЈ-ethylcarbodiimide hydrochloride (EDC), anchorage and the other side-chain protecting groups.
Oxyma, and DIPEA in anhydrous tetrahydrofuran (THF). Thus, we prepared the cyclic peptidyl resin 6, which was
The tBu group was removed with TFA/CH2Cl2 (1:1). Fmoc- then treated with 1% TFA in CH2Cl2 while stirring for
Glu(NH-CH2-CϵCH)-OH was obtained as a white powder 5 min at room temperature. This treatment was repeated
in 57% overall yield and was characterized by NMR spec- until the yellow color produced by release of the Mtt cation
troscopy and mass spectrometry. After assembly of the lin- disappeared. Following Mtt removal, the Nε-amino group
ear sequence 5, allyl and Fmoc group removal followed by was acylated with propiolic acid under standard coupling
cyclization afforded the alkynyl cyclic peptidyl resin 2. Aci- conditions. An aliquot of the resulting resin was cleaved
dolytic cleavage of an aliquot of this resin yielded c(Lys- and the crude mixture was analyzed by HPLC and charac-
Lys-Glu(NH-CH2-CϵCH)-Lys-Lys-Phe-Lys-Lys-Leu-Gln)
terized by ESI-MS. Thus, c[Lys-Lys-Lys(CO-CϵCH)-Lys-
(BPC580) in 99% HPLC purity and was characterized by Lys-Phe-Lys-Lys-Leu-Gln] (BPC468) was obtained in 99%
mass spectrometry.
purity.
Scheme 2. Synthesis of the alkynyl cyclic peptidyl resin 2.
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Cyclic Peptidotriazoles Against Phytopathogenic Bacteria
Scheme 3. Synthesis of the alkynyl cyclic peptidyl resin 3.
With the alkynyl cyclic peptidyl resins 1–3 in hand, we I (Table 1). Completion of the reactions was evaluated by
proceeded to subject them to a cycloaddition reaction with ESI/MS analysis of the crude reaction mixtures after acido-
an azide to obtain the corresponding cyclic peptidotriazoles lytic cleavage of an aliquot of the resin. Resins 1–3 were
Table 1. Synthesis of cyclic peptidotriazoles I.
[a] Reaction time: For NaN3: 2ϫ 10 h; for BnN3 and Boc-Nle(ε-N3)-OH: 1ϫ 5 h. [b] Percentage determined by HPLC analysis with
detection at 220 nm of the crude reaction mixture. [c] The crude reaction contained a mixture of BPC700 and BPC468.
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Scheme 4. Synthesis of the conjugated peptide BPC472.
treated with NaN3, BnN3, and Boc-Nleu(ε-N3)-OH pressure at room temperature. Fmoc-Nle(ε-N3)-OH was
(5 equiv.) in the presence of ascorbic acid (5 equiv.) and CuI obtained in 89% overall yield, which was characterized by
(5 equiv.) in piperidine/DMF (2:8) while stirring at room NMR and mass spectrometry. This residue was then used
temperature. Formation of the triazole moiety using BnN3 for the synthesis of BP252 (91% purity). The click reaction
was accomplished in 5 h, whereas cycloadditions with between resin 1 and the azidopentapeptide BP252 (5 equiv.)
NaN3 required two treatments of 10 h each to enable the was carried out as described previously in the presence of
reaction to reach completion. When using Boc-Nleu(ε-N3)- ascorbic acid (5 equiv.) and CuI (5 equiv.) in piperidine/
OH, the expected cyclic peptidotriazoles were obtained DMF (2:8), while stirring at room temperature for 7 h. The
from resins 1 and 2 after a 5 h treatment. In contrast, when resulting conjugated peptide BPC472 was cleaved from the
resin 3 was subjected to these conditions, the reaction did resin with TFA/H2O/TIS (95:2.5:2.5) and analyzed by
not reach completion. ESI-MS analysis of the crude reac- HPLC and mass spectrometry; the peptide was obtained in
tion mixture showed the expected cyclic peptidotriazole 98% purity.
BPC700 together with the alkynyl cyclic decapeptide
BPC468. Overnight treatment did not improve the results.
Next, cyclic peptidotriazoles were individually cleaved from
the resin with TFA/H2O/TIS (95:2.5:2.5) and analyzed by
Synthesis of Cyclic Peptidotriazoles II
HPLC and ESI-MS. Except for BPC700, cyclic peptidotri-
azoles were obtained in excellent purities (88–98%).
The synthesis of cyclic peptidotriazoles II (Figure 1) in-
volved the cycloaddition reaction of resin-bound azide 7
The above methodology was extended to the synthesis of and an alkyne in solution. The azido cyclic peptidyl resin 7
a conjugated peptide containing a cyclic and a linear pept- was prepared by following the Fmoc/tBu/All strategy pre-
idyl sequence linked through a triazole ring. We chose as viously described (Scheme 5). First, the linear peptidyl resin
the model system the reaction between the alkynyl cyclic 8 was prepared by incorporating Fmoc-Nle(ε-N3)-OH at
peptidyl resin 1 and the azido pentapeptide Fmoc-Phe- the 3-position followed by allyl and Fmoc group removal.
Nle(ε-N3)-Lys-Leu-Gln-OAll (BP252) (Scheme 4). The Subsequent cyclization under standard conditions afforded
pentapeptide BP252 was prepared by following the stan- the cyclic peptidyl resin 7. Acidolytic cleavage of an aliquot
dard protocol previously described by incorporating Fmoc- of 7 yielded BPC464 in 94% HPLC purity, which was char-
Nle(ε-N3)-OH at the 2-position. This amino acid is not acterized by mass spectrometry.
commercially available and was synthesized from Fmoc-
Resin 7 was then subjected to a cycloaddition reaction
Lys(Boc)-OH.[10] After Boc removal with TFA/CH2Cl2 with the alkynes phenylacetylene, p-ethynyltoluene and
(1:1), azidation of Fmoc-Lys-OH was performed by treat- tetrahydro-2-(prop-2-ynyloxy)-2H-pyran (5 equiv.) in the
ment with TfN3 in the presence of NaHCO3 and presence of ascorbic acid (5 equiv.) and CuI (5 equiv.) in
CuSO4·5H2O. The mixture was stirred overnight under piperidine/DMF (2:8), while stirring for 5 h at room tem-
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Cyclic Peptidotriazoles Against Phytopathogenic Bacteria
Scheme 5. Synthesis of the azido cyclic peptidyl resin 7.
Table 2. Synthesis of cyclic peptidotriazoles II.
[a] Percentage determined by HPLC analysis with detection at 220 nm of the crude reaction mixture.
perature (Table 2). After acidolytic cleavage, the corre- pv. syringae, six sequences showed MIC values of less than
sponding cyclic peptidotriazoles were obtained in excellent 12.5 μm, with BPC548 being as active as BPC194 (MIC of
purities (90–98%) and were characterized by mass spec- 3.1 to 6.2 μm). E. amylovora was the least sensitive pathogen
trometry.
to these cyclic peptidotriazoles, with three sequences exhib-
iting MIC values of less than 25 μm. Interestingly, the con-
jugated peptide BPC472 was as active as BPC194 against
X. axonopodis pv. vesicatoria (MIC of 3.1 to 6.2 μm).
In general, cyclic peptidotriazoles incorporating a tri-
azolyl lysine (BPC692, BPC696) or a triazolyl norleucine
(BPC516, BPC548, BPC552) were more active than those
bearing a triazolyl alanine (BPC456, BPC458, BPC460,
BPC472) or a triazolyl glutamine (BPC520, BPC522,
BPC532). Regarding the substituent on the triazole ring,
the introduction of a benzyl group did not significantly al-
ter the antibacterial activity. In contrast, the incorporation
of a 2-aminohexanoic group or a peptidyl chain resulted in
Biological Activity
Cyclic peptidotriazoles were tested for in vitro growth
inhibition of the plant pathogenic bacteria E. amylovora, P.
syringae pv. syringae and X. axonopodis pv. vesicatoria at
3.1, 6.2, 12.5, 25 and 50 μm (Table 3). The results were com-
pared to those obtained for BPC194. Most peptidotriazoles
were considerably active against X. axonopodis pv. vesicato-
ria and P. syringae pv. syringae. Five sequences displayed
the same MIC values as BPC194 (MIC of 3.1 to 6.2 μm)
against X. axonopodis pv. vesicatoria. Against P. syringae
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All compounds were analyzed under standard analytical high-per-
formance liquid chromatography (HPLC) conditions with a Di-
onex liquid chromatography instrument. Detection was performed
at 220 nm. Analysis was carried out with a Dionex instrument with
a Kromasil 100 C18 (40 mmϫ4.6 mm, 3.5 μm) column and a 2–
100% B linear gradient over 7 min at a flow rate of 1 mL/min (Sol-
vent A: 0.1% aq. TFA; Solvent B: 0.1% TFA in CH3CN).
Table 3. Antibacterial activity (MIC) against three plant pathogenic
bacteria and cytotoxicity of cyclic peptidotriazoles.
Peptide
MIC [μm]
Hemolysis [%][b]
Xav[a]
Ea[a]
Pss[a]
(375 μm)
BPC194
BPC456
BPC458
BPC460
BPC472
BPC516
BPC520
BPC522
BPC532
BPC548
BPC552
BPC692
BPC696
3.1–6.2
6.2–12.5
6.2–12.5
25–50
3.1–6.2
3.1–6.2
3.1–6.2
25–50
12.5–25
3.1–6.2
3.1–6.2
6.2–12.5
6.2–12.5
6.2–12.5
Ͼ50
Ͼ50
Ͼ50
Ͼ50
25–50
Ͼ50
Ͼ50
3.1–6.2
6.2–12.5
6.2–12.5
Ͼ50
17Ϯ1.7
6Ϯ0.5
5Ϯ0.2
0Ϯ0.1
6Ϯ0.3
8Ϯ0.1
2Ϯ0.2
3Ϯ0.3
4Ϯ0.4
26Ϯ4
1Ϯ1
ESI-MS analyses were performed with an Esquire 6000 ESI ion
Trap LC/MS (Bruker Daltonics) instrument equipped with an elec-
trospray ion source. The instrument was operated in the positive
ESI(+) ion mode. Samples (5 μL) were introduced into the mass
spectrometer ion source directly through an HPLC autosampler.
The mobile phase (CH3CN/H2O, 80:20; flow rate: 100 μL/min) was
delivered by a 1100 Series HPLC pump (Agilent). Nitrogen was
employed as both the drying and nebulizing gas.
Ͼ50
6.2–12.5
12.5–25
25–50
6.2–12.5
3.1–6.2
6.2–12.5
12.5–25
12.5–25
Ͼ50
12.5–25
25–50
12.5–25
12.5–25
7Ϯ0.2
30Ϯ4
HRMS were recorded under conditions of ESI with a Bruker
MicroTof-Q instrument (University of Zaragoza) or with a Bruker
MicrOTOF-Q IITM instrument (University of Girona) using a hy-
brid quadrupole time-of-flight mass spectrometer. Samples were in-
troduced into the mass spectrometer ion source directly through a
1100 Series Agilent HPLC autosampler (University of Zaragoza)
or by direct infusion through a syringe pump (University of Gi-
rona) and were externally calibrated using sodium formate. The
instruments were operated in the positive ESI(+) ion mode.
[a] Xav, Xanthomonas axonopodis pv. vesicatoria; Ea, Erwinia amyl-
ovora; Pss, Pseudomonas syringae pv. syringae. [b] Percent hemolysis
plus confidence interval (α = 0.05).
poorly active sequences (BPC460, BPC522, and BPC472).
In terms of activity against the three bacteria tested, the
best peptides were BPC516, BPC548, BPC552, BPC692,
and BPC696.
We also evaluated the toxicity to eukaryotic cells of the
cyclic peptidotriazoles; this was defined as the ability to lyse
erythrocytes in comparison to melittin. Percent hemolysis
at 375 μm is shown in Table 3. All compounds displayed low
hemolytic activity. Ten out of 12 sequences displayed less
than 10% hemolysis at 375 μm, being less hemolytic than
the parent peptide BPC194. The cyclic peptidotriazole with
an optimal balance between antibacterial and hemolytic ac-
tivities was BPC548.
1H and 13C NMR spectra were measured with a Bruker 400 MHz
NMR spectrometer. Chemical shifts are reported as δ values (ppm)
directly referenced to the solvent signal.
Fmoc-Glu(NH-CH2-CϵCH)-OtBu:
Propargylamine
(110 μL,
1.56 mmol), Oxyma (404 mg, 2.84 mmol), DIPEA (720 μL,
4.22 mmol), and EDC (320 mg, 3.12 mmol) were sequentially
added to a solution of Fmoc-Glu-OtBu (600 mg, 1.42 mmol) in
anhydrous THF (70 mL) under N2. The reaction mixture was
stirred at room temperature under N2 and monitored by HPLC.
After 24 h, further EDC (160 mg, 1.56 mmol), Oxyma (101 mg,
0.71 mmol), and DIPEA (125 μL, 0.73 mmol) were added and the
mixture was stirred for an additional 24 h. The reaction was
stopped by adding EtOH (2 mL). Removal of the solvents under
vacuum gave a residue that was dissolved in EtOAc (50 mL), ex-
tracted with H2SO4 (0.5 m; 4ϫ 50 mL), washed with distilled H2O
(2ϫ 50 mL), dried with anhydrous MgSO4, and concentrated. The
crude product was purified by column chromatography (hexane/
EtOAc, 2:1) to give Fmoc-Glu(NH-CH2-CϵCH)-OtBu as a a yel-
Conclusions
Analogues of the antimicrobial cyclic decapeptide
BPC194, bearing a 1,2,3-triazole ring at the side-chain of
the residue at the 3-position, were synthesized on solid-
phase. Formation of the triazole moiety was performed by
a copper-catalyzed cycloaddition reaction. The correspond-
ing akynyl or azido peptidyl resins were prepared and
treated with a range of azides or alkynes, respectively. Cy-
clic peptidotriazoles were obtained in excellent purities, and
sequences with high antibacterial and low hemolysis were
identified. The best cyclic peptidotriazole, BPC548, dis-
played a biological activity profile similar to that of the par-
ent peptide. These results offer a good perspective for the
further development of new antimicrobial agents.
low oil (639 mg, 98% yield). Rf = 0.81 (hexane/EtOAc, 1:5); tR
=
8.94 min. IR (neat): ν = 3305.99 (ϵCH, st), 2980.46 (CϵC, st),
˜
1725.79, 1689.73 (C=O, st), 1637.08 (δ NH2), 1083.99 (C–O, st),
738.99 (δ NH, opp), 646.23 (δϵCH) cm–1. 1H NMR (400 MHz,
CDCl3): δ = 1.51 [s, 9 H, C(CH3)3], 1.90–1.97 [m, 2 H, CH2(β),
CH2(γ)], 2.23–2.30 [m, 3 H, CH2(β), CH2(γ), CϵCH], 4.07–4.09
(m, 2 H, NCH2), 4.24–4.27 [m, 2 H, CHFmoc, CH(α)], 4.42–4.51
(m, 2 H, OCH2), 5.59 (d, J = 7.6 Hz, 1 H, NHCOO), 6.32 (br., 1
H, CONH), 7.36 (t, J = 7.6 Hz, 2 H, 2 CHAr), 7.45 (t, J = 7.6 Hz,
2 H, 2 CHAr), 7.63 (d, J = 7.6 Hz, 2 H, 2 CHAr), 7.81 (d, J =
7.6 Hz, 2 H, 2 CHAr) ppm. 13C NMR (100 MHz, CDCl3): δ = 27.92
(CH3), 29.06, 29.19, 32.23 [CH2(β), CH2(γ), CH2N], 47.15
(CHFmoc), 53.80 [CH(α)], 66.93 (OCH2), 71.52 (ϵCH), 79.41 (Cϵ),
82.63 [C(CH3)3], 119.94 (CHAr), 119.97 (CHAr), 125.01 (CHAr),
125.06 (CHAr), 127.04 (2 CHAr), 127.70 (2 CHAr), 141.25 (CAr),
141.29 (CAr), 143.57 (CAr), 143.81 (CAr), 156.42 (NHCOO), 170.96
(COOtBu), 171.71 (CONH) ppm. MS (ESI): m/z = 463.1 [M +
H]+, 485.1 [M + Na]+.
Experimental Section
Commercially available reagents were used throughout without pu-
rification. Solvents were purified and dried by passing them
through an activated alumina purification system (MBraun SPS-
800) or by conventional distillation techniques. Flash chromatog-
raphy purifications were performed on silica gel 60 (230–400 mesh,
Merck).
Fmoc-Glu(NH-CH2-CϵCH)-OH: Fmoc-Glu(NH-CH2-CϵCH)-
OtBu (400 mg, 0.71 mmol) was dissolved in a solution of TFA/
8
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Date: 26-06-13 17:22:24
Pages: 12
Cyclic Peptidotriazoles Against Phytopathogenic Bacteria
CH2Cl2 (1:1, 10 mL) and stirred for 2 h at room temperature. Fol- 94% yield). Rf = 0.64 (CH2Cl2/MeOH, 7:1); tR = 8.60 min. IR
lowing TFA evaporation and diethyl ether extraction, Fmoc-
Glu(NH-CH2-CϵCH)-OH was obtained as white powder
(81 mg, 58% yield). Rf = 0.32 (EtOAc/NH3/MeOH, 5:1:1); tR
(neat): ν = 3381.58 (ϵCH, st), 2095.48 (NϵN, st), 1701.11 (C=O,
˜
a
st), 1521.57 (C=C, st), 1450.02 (δ CH2), 1190.06 (δ CH, ip), 739.37
=
(δ
NH,
opp) cm–1.
1H
NMR
(400 MHz,
[D6]-
7.70 min. IR (neat): ν = 3293.84 (ϵCH, st), 2923.38 (CϵC, st),
DMSO): δ = 1.34–1.43 [m, 2 H, 2 CH2(γ)], 1.45–1.57 [m, 2 H,
CH2(δ)], 1.58–1.66 [m, 1 H, CH2(β)], 1.68–1.77 [m, 1 H, CH2(β)],
˜
1687.41 (C=O, st), 1640.36 (δ NH2), 1536.22, 1448.67 (δ CH2),
1
1085.73 (C–O, st), 738.22 (δ NH, opp), 620.39 (δ ϵCH) cm–1. H 3.32 [t, J = 6.8 Hz, 2 H, CH2(ε)], 3.91–3.97 [m, 1 H, CH(α)], 4.21–
NMR (400 MHz, CDCl3): δ = 1.91–1.98 [m, 1 H, CH2(β)], 2.12– 4.25 [m, 1 H, CH(Fmoc)], 4.28–4.30 [m, 2 H, CH2(Fmoc)], 7.42 (td, J
2.24 [m, 1 H, CH2(β)], 2.28–2.33 [m, 2 H, CH2(γ)], 2.56 (t, J =
3.6 Hz, 1 H, ϵCH), 3.93 (d, J = 3.6 Hz, 2 H, NCH2), 4.15–4.22
[m, 2 H, CHFmoc, CH(α)], 4.31 (dd, J = 9.4, 13.8 Hz, 1 H, OCH2),
= 0.9, 7.4 Hz, 2 H, 2 CHAr), 7.42 (t, J = 7.4 Hz, 2 H, 2 CHAr),
7.65 (d, J = 7.4 Hz, 1 H, CHAr), 7.73 (d, J = 7.4 Hz, 1 H, CHAr),
7.89 (d, J = 7.4 Hz, 2 H, 2 CHAr), 8.95 (s, 1 H, CONH) ppm. 13C
4.37 (dd, J = 9.4, 13.8 Hz, 1 H, OCH2), 7.30 (td, J = 1.6, 9.8 Hz, NMR (100 MHz, [D6]DMSO): δ = 23.37 [CH2(γ)], 28.29 [CH2(δ)],
2 H, 2 CHAr), 7.38 (t, J = 9.8 Hz, 2 H, 2 CHAr), 7.64–7.68 (m, 2 30.74 [CH2(β)], 47.13 [CH(Fmoc)], 50.98 [CH2(ε)], 54.12 [CH(α)],
H, 2 CHAr), 7.77 (d, J = 9.8 Hz, 2 H, 2 CHAr) ppm. 13C NMR
66.05 [CH2(Fmoc)], 120.56 (CHAr), 120.58 (CHAr), 125.73 (CHAr),
(100 MHz, CDCl3): δ = 28.54 (CH2-β), 29.45 (NCH2), 33.05 (CH2- 125.76 (CHAr), 127.52 (2 CHAr), 128.10 (2 CHAr), 141.20 (CAr),
γ), 46.99 (CHFmoc), 54.91 (CH-α), 67.96 (OCH2), 72.18 (ϵCH), 141.22 (CAr), 144.27 (CAr), 144.32 (CAr), 156.63 (CONH), 174.32
80.51 (ϵC), 120.86 (2 CHAr), 126.23 (2 CHAr), 128.13 (2 CHAr), (COOH) ppm. MS (ESI): m/z = 395.1 [M + H]+. HRMS (ESI):
128.74 (2 CHAr), 142.51 (2 CAr), 145.10 (CAr), 145.27 (CAr), 158.62 calcd. for C21H23N4O4 395.1714; found 395.1727 and calcd. for
(NHCOO), 174.46 (CONH), 175.35 (COOH) ppm. MS (ESI): m/z C21H22N4NaO4 417.1533; found 417.1544.
= 407.0 [M + H]+. HRMS (ESI): calcd. for C23H23N2O5 407.1601;
found 407.1591 and calcd. for C23H22N2NaO5 429.1421; found
429.1406.
General Method for the Synthesis of Alkynyl and Azido Linear Pept-
idyl Resins: These peptidyl resins were synthesized manually by the
solid-phase method using standard Fmoc chemistry. Fmoc-Rink-
MBHA resin (0.3 mmol/g) was used as solid support. Fmoc-Leu-
OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Nle(ε-N3)-
OH, Fmoc-Prg-OH, Fmoc-Glu(NH-CH2-CϵCH)-OH, Fmoc-Phe-
OH or Fmoc-Glu-OAll were used as amino acid derivatives. Pept-
ide elongation was performed by repeated cycles of Fmoc group
removal, coupling, and washings. Fmoc group removal was
achieved with piperidine/DMF (3:7, 2 + 10 min). Couplings of the
Fmoc-amino acids (4 equiv.) were mediated by Oxyma (4 equiv.)
and DIPCDI (4 equiv.) in DMF at room temperature for 1 h while
stirring. The completion of the reactions was checked by the Kaiser
test.[11] After each coupling and deprotection step, the resin was
washed with DMF (6ϫ 1 min) and CH2Cl2 (6ϫ 1 min), and air-
dried. After the fifth coupling, NMP was used instead of DMF.
An aliquot of each resulting peptidyl resin was treated with TFA/
H2O/triisopropylsilane (TIS) (95:2.5:2.5) for 2 h at room tempera-
ture. Following TFA evaporation and diethyl ether extraction, the
crude peptide was dissolved in H2O, lyophilized, analyzed by
HPLC, and characterized by mass spectrometry.
Fmoc-Lys-OH: Fmoc-Lys(Boc)-OH (1.25 g, 3.40 mmol) was dis-
solved in a solution of TFA/CH2Cl2 (1:1, 75 mL) and stirred for
2 h at room temperature. Following TFA evaporation and diethyl
ether extraction, Fmoc-Lys-OH was obtained as a white powder
(1.22 g, 95% yield). Rf = 0.5 (CHCl3/MeOH/AcOH, 5:3:1); tR
=
6.99 min. IR (neat): ν = 3066.47 (ϵCH, st), 1670.64 (C=O, st),
˜
1519.64 (C=C), 1449.63 (δ CH2), 1181.57 (δ C–H, ip), 789.19 (γ
CH2), 738.60 (δ NH, opp) cm–1. 1H NMR (400 MHz, [D6]DMSO):
δ = 1.344–1.430 [m, 2 H, 2 CH2(γ)], 1.48–1.71 [m, 4 H, 2 CH2(β),
2 CH2(δ)], 2.73–2.80 [m, 2 H, 2 CH2(ε)], 3.90–3.96 [m, 1 H, CH(α)],
4.21–4.26 [m, 1 H, CH(Fmoc)], 4.28–4.34 [m, 2 H, CH2(Fmoc)], 7.33
(td, J = 0.7 7.2 Hz, 2 H, 2 CHAr), 7.42 (t, J = 7.2 Hz, 2 H, 2 CHAr),
7.63 (d, J = 7.2 Hz, 1 H, CHAr), 7.71–7.74 (m, 1 H, CHAr), 7.79
(br., 1 H, CONH), 7.89 (d, J = 7.2 Hz, 2 H, 2 CHAr) ppm. 13C
NMR (100 MHz, [D6]DMSO): δ = 22.57 [CH2(γ)], 26.51 [CH2(β)],
30.17 [CH2(δ)], 38.59 [CH2(ε)], 46.66 [CH(Fmoc)], 53.61 [CH(α)],
65.58 [CH2(Fmoc)], 120.13 (CHAr), 120.14 (CHAr), 125.26 (2 CHAr),
127.07 (CHAr), 127.08 (CHAr), 127.65 (2 CHAr), 140.73 (CAr),
140.76 (CAr), 143.79 (CAr), 143.81 (CAr), 156.20 (CONH), 173.85
(COOH) ppm. MS (ESI): m/z = 369.1 [M + H]+.
Fmoc-Lys(Boc)-Lys(Boc)-Prg-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Glu(Rink-MBHA)-OAll (4): Synthesized following
the procedure described above incorporating Fmoc-Prg-OH at the
3-position. After acidolytic cleavage of an aliquot of this resin,
Fmoc-Lys-Lys-Prg-Lys-Lys-Phe-Lys-Lys-Leu-Gln-OAll was ob-
tained in 85% purity. tR = 6.24 min. MS (ESI): m/z = 767.5 [M +
2H]2+, 1534.1 [M + H]+.
Fmoc-Nle(ε-N3)-OH:[10] NaN3 (883 mg, 13.58 mmol) was dissolved
in a mixture of distilled H2O (2 mL) and CH2Cl2 (3.5 mL). Triflic
anhydride (Tf2O) (460 μL, 2.72 mmol) was added slowly, and the
resulting mixture was stirred for 2 h. The organic phase was re-
moved and the aqueous phase was extracted with CH2Cl2 (2ϫ
3 mL). The organic fractions containing TfN3 were combined,
washed with a saturated aqueous solution of Na2CO3 (6.5 mL),
and used without further purification.
Fmoc-Lys(Boc)-Lys(Boc)-Glu(NH-CH2 -CϵCH)-Lys-
(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)-
OAll (5): Synthesized following the procedure described above in-
corporating Fmoc-Glu(NH-CH2-CϵCH)-OH at the 3-position.
After acidolytic cleavage of an aliquot of this resin, Fmoc-Lys-Lys-
Glu(NH-CH2-CϵCH)-Lys-Lys-Phe-Lys-Lys-Leu-Gln-OAll was
obtained in 91% purity. tR = 6.41 min. MS (ESI): m/z = 802.5 [M
+ 2H]2+, 1605.1 [M + H]+.
Fmoc-Lys-OH (500 mg, 1.36 mmol) was dissolved in distilled H2O
(4.5 mL) and MeOH (9 mL). Thereafter, NaHCO3 (1.14 g,
13.58 mmol) and CuSO4·5H2O (34 mg, 13 mmol) were added.
TfN3 in CH2Cl2 (9.5 mL) was then added and the mixture was
stirred under pressure at room temperature. The reaction was moni-
tored by HPLC. After 12 h, the organic solvents were removed un-
der vacuum, and the remaining solution was diluted with distilled
H2O (36 mL) and acidified to pH 2 by the addition of aq. HCl.
After extraction with EtOAc (4ϫ 20 mL), the organic fractions
were combined, washed with brine (20 mL), dried with anhydrous
MgSO4, and concentrated. The crude product was digested with
pentane to give Fmoc-Nle(ε-N3)-OH as a white powder (385 mg,
Fmoc-Lys(Boc)-Lys(Boc)-Lys(Mtt)-Lys(Boc)-Lys(Boc)-Phe-Lys-
(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)-OAll: Synthesized follow-
ing the procedure described above incorporating Fmoc-Lys(Mtt)-
OH at the 3-position. After acidolytic cleavage of an aliquot of this
resin, Fmoc-Lys-Lys-Lys-Lys-Lys-Phe-Lys-Lys-Leu-Gln-OAll was
obtained in 94% purity. tR = 6.01 min. MS (ESI): m/z = 784.0 [M
+ 2H]2+, 1566.0 [M + H]+, 1588.0 [M + Na]+.
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Planas, L. Feliu et al.
FULL PAPER
F m o c - Ly s ( B o c ) - Ly s ( B o c ) - N l e ( ε - N 3 ) - Ly s ( B o c ) - Ly s - above for the coupling of Fmoc-protected amino acids. After acido-
(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)-OAll (8): lytic cleavage of an aliquot of the resulting resin 3, c[Lys-Lys-Lys-
Synthesized following the procedure described above incorporating
Fmoc-Nle(ε-N3)-OH at the 3-position. After acidolytic cleavage of
an aliquot of this resin, Fmoc-Lys-Lys-Nle(ε-N3)-Lys-Lys-Phe-
Lys-Lys-Leu-Gln-OAll was obtained in 77% purity. tR = 6.61 min.
MS (ESI): m/z = 1592.2 [M + H]+.
(CO-CϵCH)-Lys-Lys-Phe-Lys-Lys-Leu-Gln] (BPC468) was ob-
tained in 99% purity. tR = 5.98 min. MS (ESI): m/z = 1337.7 [M +
H]+, 1359.7 [M + Na]+.
c[Lys(Boc)-Lys(Boc)-Nle(ε-N3)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Glu(Rink-MBHA)] (7): Synthesized from resin 8 fol-
lowing the procedure described above. After acidolytic cleavage of
an aliquot of resin 7, c[Lys-Lys-Nle(ε-N3)-Lys-Lys-Phe-Lys-Lys-
Leu-Gln] (BPC464) was obtained in 94% purity. tR = 6.00 min. MS
(ESI): m/z = 1312.0 [M + H]+, 1334.0 [M + Na]+.
Synthesis of Fmoc-Phe-Nle(ε-N3)-Lys-Leu-Gln-OAll (BP252): The
peptidyl resin Fmoc-Phe-Nle(ε-N3)-Lys(Boc)-Leu-Glu(Rink-
MBHA)-OAll was prepared following the general procedure de-
scribed above for the synthesis of linear azido peptidyl resins. Aci-
dolytic cleavage afforded Fmoc-Phe-Nle(ε-N3)-Lys-Leu-Gln-OAll
(BP252) in 91% purity. tR = 8.29 min. MS (ESI): m/z = 951.4 [M
+ H]+.
Synthesis of the Cyclic Peptidotriazoles: The corresponding alkynyl
or azido cyclic peptidyl resin was swollen with CH2Cl2 (1ϫ 20 min)
and DMF (1ϫ 20 min) and then treated with an azide (5 equiv.)
or with an alkyne (5 equiv.), respectively, in the presence of ascorbic
acid (5 equiv.) and CuI (5 equiv.) in piperidine/DMF (2:8). The re-
action mixture was stirred for 5 h at room temperature. The resin
was subsequently washed with sodium N,N-diethyldithiocarbamate
(0.03 m in NMP, 3ϫ 3 min), DMF (6ϫ 1 min) and CH2Cl2 (1ϫ
20 min). The resulting cyclic peptidotriazole was cleaved from the
resin with TFA/H2O/TIS (95:2.5:2.5) and analyzed by HPLC and
mass spectrometry.
General Method for the Synthesis of Alkynyl and Azido Cyclic Pept-
idyl Resins 1–3 and 7: The C-terminal allyl ester of the correspond-
ing linear peptidyl resin was cleaved by treatment with [Pd(PPh3)4]
(3 equiv.) in CHCl3/AcOH/NMM (3:2:1) under nitrogen and stir-
ring for 3 h at room temperature. After this time, the resin was
washed with THF (3ϫ 2 min), NMP (3ϫ 2 min), DIPEA/CH2Cl2
(1:19, 3ϫ 2 min), sodium N,N-diethyldithiocarbamate (0.03 m in
NMP, 3ϫ 15 min), NMP (10ϫ 1 min) and CH2Cl2 (3ϫ 2 min).
Fmoc was removed with piperidine/DMF (3:7, 2 + 10 min) fol-
lowed by washes with DMF (6ϫ 1 min) and CH2Cl2 (3ϫ 1 min).
Cyclization was carried out by treating the resulting resin with
[ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphon-
ium hexafluorophosphate (PyOxim) (5 equiv.), Oxyma (5 equiv.),
and DIPEA (10 equiv.) in NMP, while stirring for 24 h. Following
washes with NMP (6ϫ 1 min) and CH2Cl2 (6ϫ 1 min), an aliquot
of the cyclic peptidyl resin was cleaved by treatment with TFA/
H2O/TIS (95:2.5:2.5) for 2 h. Following TFA evaporation and di-
ethyl ether extraction, the crude peptide was dissolved in H2O, lyo-
philized, analyzed by HPLC, and characterized by mass spectrome-
try.
Cyclic Peptidotriazole BPC456: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Prg-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu-
(Rink-MBHA)] (1) was treated with NaN3 following the general
procedure described above. In this case, two treatments of 10 h were
required. BPC456 was obtained in 97% purity. tR = 5.87 min. MS
(ESI): m/z = 1295.7 [M + H]+ . HRMS (ESI): calcd. for
C61H109N20O11 432.6189; found 432.6211 and calcd. for
C61H108N20O11 648.4248; found 648.4263.
Cyclic Peptidotriazole BPC458: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Prg-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu-
(Rink-MBHA)] (1) was treated with BnN3 for 5 h following the
general procedure described above. BPC458 was obtained in 94%
purity. tR = 5.98 min. MS (ESI): m/z = 1385.7 [M + H]+, 1426.7
[M + K]+. HRMS (ESI): calcd. for C68H115N20O11 462.6346; found
462.6365 and calcd. for C68H114N20O11 693.4482; found 693.4478.
c[Lys(Boc)-Lys(Boc)-Prg-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (1): Synthesized from resin 4 follow-
ing the procedure described above. After acidolytic cleavage of an
aliquot of resin 1, c(Lys-Lys-Prg-Lys-Lys-Phe-Lys-Lys-Leu-Gln)
(BPC466) was obtained in 96% purity. tR = 5.81 min. MS (ESI):
m/z = 1253.0 [M + H]+, 1274.9 [M + Na]+. HRMS (ESI): calcd.
for C61H108N17O11 418.2799; found 418.2794 and calcd. for
C61H107N17O11 626.9163; found 626.9150.
Cyclic Peptidotriazole BPC460: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Prg-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu-
(Rink-MBHA)] (1) was treated with Boc-Nle(ε-N3)-OH for 5 h fol-
lowing the general procedure described above. BPC460 was ob-
tained in 90% purity. tR = 5.85 min. MS (ESI): m/z = 1424.7 [M
+ H]+. HRMS (ESI): calcd. for C67H120N21O13 475.6453; found
475.6464 and calcd. for C67H119N21O13 712.9643; found 712.9649.
c[Lys(Boc)-Lys(Boc)-Glu(NH-CH2-CϵCH)-Lys(Boc)-Lys(Boc)-
Phe-Lys(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)] (2): Synthesized
from resin 5 following the procedure described above. After acido-
lytic cleavage of an aliquot of resin 2, c[Lys-Lys-Glu(NH-CH2-
CϵCH)-Lys-Lys-Phe-Lys-Lys-Leu-Gln] (BPC580) was obtained in
99% purity. tR = 6.08 min. MS (ESI): m/z = 1324.0 [M + H]+,
1346.0 [M + Na]+.
Cyclic Peptidotriazole BPC472: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Prg-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu-
(Rink-MBHA)] (1) was treated with Fmoc-Phe-Nle(ε-N3)-Lys-
Leu-Gln-OH (BP252) for 7 h following the general procedure de-
scribed above. BPC472 was obtained in 98% purity. tR = 6.15 min.
MS (ESI): m/z = 1981.6 [M + H]+. HRMS (ESI): calcd. for
C9 6 H1 6 5 N2 7 O1 8 496.0701; found 496.3234; calcd. for
C96H164N27O18 661.0910; found 661.4269 and calcd. for
C96H163N27O18 991.1329; found 991.6358.
c(Lys(Boc)-Lys(Boc)-Lys(CO-CϵCH)-Lys(Boc)-Lys(Boc)-Phe-Lys-
(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)) (3): Peptidyl resin c[Lys-
(Boc)-Lys(Boc)-Lys(Mtt)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (6) was prepared following the gene-
ral procedure described above from Fmoc-Lys(Boc)-Lys(Boc)-
Lys(Mtt)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu-
(Rink-MBHA)-OAll. Peptidyl resin 6 was treated with 1% TFA in
CH2Cl2 (the solution became yellow). The mixture was stirred for
5 min at room temperature and the resin was then washed with
CH2Cl2 (2ϫ 1 min), MeOH (2ϫ 1 min) and CH2Cl2 (2ϫ 1 min).
TFA treatment was repeated until the solution remained colorless.
The Nε-amino group of the lysine residue at the 3-position was
then acylated with propiolic acid using the conditions described
Cyclic Peptidotriazole BPC516: The azido peptidyl resin c[Lys-
(Boc)-Lys(Boc)-Nle(ε-N3)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (7) was treated with phenylacetylene
for 5 h following the general procedure described above. BPC516
was obtained in 98% purity. tR = 6.09 min. MS (ESI): m/z = 1414.0
[M + H]+ , 1436.0 [M + Na]+ . HRMS (ESI): calcd. for
C70H119N20O11 471.9784; found 471.9808 and calcd. for
C70H118N20O11 707.4639; found 707.4654.
10
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O30215
/KAP1
Date: 26-06-13 17:22:24
Pages: 12
Cyclic Peptidotriazoles Against Phytopathogenic Bacteria
Cyclic Peptidotriazole BPC520: The alkynyl resin c[Lys(Boc)-Lys-
PMV6076 (Institut National de la Recherche Agronomique, Ang-
(Boc)-Glu(NH-CH2-CϵCH)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)- ers, France), Pseudomonas syringae pv. syringae EPS94 (Institut de
Lys(Boc)-Leu-Glu(Rink-MBHA)] (2) was treated with BnN3 for Tecnologia Agroalimentària, Universitat de Girona, Spain), and
5 h following the general procedure described above. BPC520 was
obtained in 98% purity. tR = 6.04 min. MS (ESI): m/z = 1457.0 [M
+ H]+, 1479.0 [M + Na]+. HRMS (ESI): calcd. for C71H120N21O12
486.3136; found 486.3157 and calcd. for C71H119N21O12 728.9668;
found 728.9677.
Xanthomonas axonopodis pv. vesicatoria 2133–2 (Instituto Valenci-
ano de Investigaciones Agrarias, Valencia, Spain). All bacteria were
stored in Luria Bertani (LB) broth supplemented with glycerol
(20%) and maintained at –80 °C. E. amylovora and P. syringae pv.
syringae were scrapped from LB agar after growing for 24 h and
X. axonopodis pv. vesicatoria after growing for 48 h at 25 °C. The
cell material was suspended in sterile water to obtain a suspension
of 108 CFU mL–1.
Cyclic Peptidotriazole BPC522: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Glu(NH-CH2-CϵCH)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Glu(Rink-MBHA)] (2) was treated with Boc-Nle(ε-
N3)-OH for 5 h following the general procedure described above.
BPC522 was obtained in 96% purity. tR = 5.66 min. MS (ESI): m/z
= 1496.1 [M + H]+, 1518.1 [M + Na]+. HRMS (ESI): calcd. for
Antibacterial Activity: Lyophilized compounds were solubilized in
sterile Milli-Q water to a final concentration of 1000 μm and filter
sterilized through a 0.22 μm pore filter. For minimum inhibitory
C
70H125N22O14 499.3243; found 499.3276 and calcd. for concentration (MIC) assessment, dilutions of the compounds were
C70H124N22O14 748.4828; found 748.4837.
made to obtain a stock concentration of 500, 250, 125, 62.5 and
31.125 μm. Aliquots (20 μL) of each dilution were mixed in a micro-
titer plate well with the corresponding suspension of the bacterial
indicator (20 μL), Trypticase Soy Broth (TSB) (BioMèrieux,
France) (160 μL) to a total volume of 200 μL. Three replicates for
each strain, compound, and concentration were used. Positive con-
trols contained water instead of compound and negative controls
contained compounds without bacterial suspension. Microbial
growth was automatically determined by optical density measure-
ment at 600 nm (Bioscreen C, Labsystem, Helsinki, Finland).
Microplates were incubated at 25 °C with 20 sec shaking before
hourly absorbance measurement for 48 h. The experiment was re-
peated twice. The MIC was taken as the lowest compound concen-
tration with no growth at the end of the experiment.
Cyclic Peptidotriazole BPC532: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Glu(NH-CH2-CϵCH)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Glu(Rink-MBHA)] (2) was treated with NaN3 fol-
lowing the general procedure described above. In this case, two
treatments of 10 h were required. BPC532 was obtained in 98%
purity. tR = 5.79 min. MS (ESI): m/z = 1367.0 [M + H]+, 1388.9 [M
+ Na]+. HRMS (ESI): calcd. for C64H114N21O12 456.3047; found
456.3005 and calcd. for C64H113N21O12 683.9433; found 683.9449.
Cyclic Peptidotriazole BPC548: The azido peptidyl resin c[Lys-
(Boc)-Lys(Boc)-Nle(ε-N3)-Lys(Boc)-Lys(Boc)-Phe-Lys-
(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)] (7) was treated with p-eth-
ynyltoluene for 5 h following the general procedure described
above. BPC548 was obtained in 90% purity. tR = 6.35 min. MS
(ESI): m/z = 1428.2 [M + H]+, 1450.1 [M + Na]+. HRMS (ESI):
calcd. for C71H121N20O11 476.6502; found 476.6542 and calcd. for
C71H120N20O11 714.4717; found 714.4729.
Hemolytic Activity: The hemolytic activity of the compounds was
evaluated by determining hemoglobin release from erythrocyte sus-
pensions of fresh human blood (5% vol/vol). Blood was aseptically
collected using a BD vacutainer K2E System with EDTA (Belliver
Industrial State, Plymouth, U. K.) and stored for less than 2 h at
4 °C. Blood was centrifuged at 6000 g for 5 min, washed three times
with TRIS buffer (10 mm TRIS, 150 mm NaCl, pH 7.2) and di-
luted. Compounds were solubilized in TRIS buffer to a stock con-
centration of 750, 500, and 300 μm (final concentrations tested were
375, 250 and 150 μm). Human red blood cells (65 μL) were mixed
with the compound solution (65 μL) and incubated under con-
tinuous shaking for 1 h at 37 °C. The tubes were then centrifuged
at 3500 g for 10 min. Aliquots (80 μL) of the supernatant were
transferred to 100-well microplates (Bioscreen) and diluted with
Milli-Q water (80 μL). Hemolysis was measured as the absorbance
at 540 nm with a Bioscreen plate reader. Complete hemolysis was
determined in TRIS buffer plus melittin at 100 μm final concentra-
tion (Sigma–Aldrich Corporation, Madrid, Spain) as a positive
control. The percentage of hemolysis (H) was calculated by using
the equation: H = 100ϫ[(Op–Ob)/(Om–Ob)], where Op is the den-
sity for a given compound concentration, Ob for the buffer, and
Om for the melittin positive control.
Cyclic Peptidotriazole BPC552: The azido peptidyl resin c[Lys-
(Boc)-Lys(Boc)-Nle(ε-N3)-Lys(Boc)-Lys(Boc)-Phe-Lys-
(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)] (7) was treated with tetra-
hydro-2-(prop-2-ynyloxy)-2H-pyran for 5 h following the general
procedure described above. BPC552 was obtained in 90% purity.
tR = 5.77 min. MS (ESI): m/z = 1368.1 [M + H]+, 1386.1 [M +
NH4]+, 1408.1 [M + K]+. HRMS (ESI): calcd. for C65H117N20O12
456.6381; found 456.6401 and calcd. for C65H116N20O12 684.4535;
found 684.4579.
Cyclic Peptidotriazole BPC692: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Lys(CO-CϵCH)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (3) was treated with NaN3 following
the general procedure described above. In this case, two treatments
of 10 h were required. BPC692 was obtained in 92% purity. tR
=
6.10 min. MS (ESI): m/z = 1380.8 [M + H]+, 1402.8 [M + Na]+.
HRMS (ESI): calcd. for C65H117N21O12 345.9792; found 345.9805,
calcd. for C65H116N21O12 460.9699; found 460.9706; and calcd. for
C65H115N21O13 690.9512; found 690.9513.
Supporting Information (see footnote on the first page of this arti-
Cyclic Peptidotriazole BPC696: The alkynyl resin c[Lys(Boc)-Lys-
(Boc)-Lys(CO-CϵCH)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (3) was treated with BnN3 for 5 h
following the general procedure described above. BPC696 was ob-
tained in 88% purity. tR = 6.47 min. MS (ESI): m/z = 1470.9 [M +
H]+, 1492.9 [M + Na]+. HRMS (ESI): calcd. for C72H123N21O13
368.4910; found 368.4919, calcd. for C72H122N21O13 490.9855;
found 490.9861, and calcd. for C72H121N21O13 736.4762; found
736.4739.
1
cle): Copies of HPLC, ESI-MS, IR, H and 13C NMR spectra of
amino acid derivatives. Copies of HPLC and ESI-MS of alkynyl
and azido linear and cyclic peptides. Copies of HPLC, ESI-MS and
HRMS (ESI) of cyclic peptidotriazoles.
Acknowledgments
I. G. and S. V. were the recipients of a predoctoral fellowship from
the Generalitat de Catalunya. This work was financed by MICINN
of Spain (grant numbers AGL2009-13255-C02-02/AGR and
Bacterial Strains and Growth Conditions: The following plant
pathogenic bacterial strains were used: Erwinia amylovora
Eur. J. Org. Chem. 0000, 0–0
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11

Job/Unit: O30215
/KAP1
Date: 26-06-13 17:22:24
Pages: 12
M. Planas, L. Feliu et al.
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Received: February 8, 2013
Published Online: ᭿
12
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